PROJECT SUMMARY (BRIAN SANDERS) Discovering and Engineering Hole Tunneling Pathways in Cytochrome P450BM3 as Protective Mechanisms Against Oxidative Damage Much of the life on Earth requires molecular oxygen (O2) for survival. The reason for this is that O2 is utilized in a multitude of enzymatic processes in which it is activated at a metal center such as Fe or Cu, and reduced to water, a process which drives ATP synthesis; similarly, other enzymatic opportunities exist in which O2 can be activated to form high-valent metal-oxo species that serve as potent oxidants for the incorporation of O into biomolecules. Cytochrome P450 enzymes contain an Fe-porphyrin (heme) that binds O2 and reduces it by two electrons to form one equivalent of water and a highly oxidized (Por?+)Fe(IV)=O, termed compound I (CMPD-I). This highly reactive intermediate has been implicated as the active oxidant in the catalytic cycle of P450. Interestingly, many P450 enzymes have been shown to consume O2 and reducing equivalents without concomitant substrate oxidation. Given the high frequency of turnover, this implies that CMPD-I is formed and then safely quenched without damage to the enzyme. In principle the high potential of CMPD-I (~1 V vs. NHE) should oxidatively damage the active site or the heme, thus causing loss of activity. This is an inherent but necessary danger that Nature has managed to balance delicately. Recently, Gray and Winkler proposed that chains of redox active amino acids such as tyrosine (Tyr) and tryptophan (Trp) serve to propagate oxidizing equivalents, holes, away from the active site and to the protein surface where they can be safely quenched by native reducing agents. In this project, we aim to demonstrate that protective mechanisms exist in cytochrome P450BM3. Two possible routes for hole propagation exist in P450BM3, Pathway I (P-I: Trp96, Trp90, Tyr334) and Pathway II (P-II: Cys156, Tyr115, Met112, Tyr305). We will examine the photooxidation of P450BM3 through conjugation of Ru- photosensitizer and flash-quench time-resolved spectroscopy involving the heme domain of P450BM3 in which we can photochemically inject a whole in to the active site and monitor the resulting processes. Additionally, the holo-protein will be examined under enzymatic turnover conditions. Once the experimental parameters for these methods have been established, we will make site-specific mutations to probe the role of amino acids local to the active site. First, we will test our hypothesis that Trp96, a highly-conserved residue in nearly 75% of all P450s, serves as the initial gateway for hole tunneling out of the active site. This will be accomplished by Trp96His mutation which maintains a critical H-bond but removes redox activity. Second, we will examine the effect of introducing non-native Tyr/Trp in place of phenylalanine residues around the active site. We expect that these mutations can be engineered such that hole propagation can be controlled and/or redirected. This type of electronic control will provide, (1) distance criteria for identifying redox active chains in other proteins; (2) a deeper understanding of native enzymatic protective mechanisms against oxidation; and (3) engineered biocatalysts with greater stability toward oxidative damage.